Compbead

Absolute γδ T cell counts were performed on 100 μl of fresh PB stained with following anti-human monoclonal antibodies (mAbs): CD3 (SK7; BV605) and CD45 (H130; AF700) (BioLegend) and Vδ2 (IMMU-389; FITC) (Beckman Coulter). We then used CountBright™ Absolute Counting Beads (Invitrogen) according to the manufacturer’s instructions.
For both regular and intracellular staining, γδ T cells were first screened for viability with Zombie Aqua™ Fixable Viability kit (BioLegend) and then processed as previously described [20 (link)]. The following mAbs were used: CD28 (CD28.2; PE-Cy7) (BioLegend); Vδ2 (B6; BUV395), CD3 (UCHT1; BUV661), CD45RA (H100; BUV737), CD16 (348; BUV496) (BD); CD57 (REA769; PE-Vio615) (Miltenyi); CD27 (0322; APCeFluor780) (eBioscience). The intracellular amounts of TNF-α (Mab11; PE) and IFN-γ (B27; Bv711) (BD) as well as the frequency of cytotoxic CD107a^pos cells (H4A3, PE) (BD Biosciences) was evaluated after stimulating γδ T cells with Phorbol myristate acetate (PMA; 0.5 μg/mL) and Ionomycin (0.1 μg/mL) (Sigma Aldrich).
Flow cytometry experiments were performed on FACS Symphony™ (BD). All data and t-SNE algorithm were analyzed with FlowJo Software (version 9.6) (FlowJo LLC) using single stained controls BD CompBeads™ (BD).

The stimulated and unstimulated MD-DCs were harvested from the 6-well plates using PBS/2 mM EDTA. They were then washed in PBS and stained for 30 min at 4 °C with the following monoclonal antibodies (mAbs, Becton Dickinson, Warsaw, Poland): fluorescein isothiocyanate (FITC)-conjugated anti-CD86, anti-CD40, anti-HLA-DR, anti-CD40 and anti-DC-SIGN mAbs; phycoerythrin (PE)-conjugated anti-CD80 mAb; or relevant isotype-matched mAbs as controls. Living cells were gated using forward and side scatter properties (FSC/SSC) and then using the specific markers indicated above. Data were analyzed using the FACS LSRII (BD) and FlowJo software. Compensations were calculated using BD Compbeads with the automatic compensation program. Data were expressed as percentages of cells expressing the marker and the mean fluorescence intensities (MFI), representing the molecular densities on the cell surface for each marker for the considered population, after subtraction of the isotype control.

Flow cytometry was performed on a Becton Dickinson LSRFortessa X-20 flow cytometer, utilizing violet (405 nM), blue (488 nM), yellow–green (561 nM), and red (640 nM) lasers. Compensation settings were conducted using BD CompBeads (BD Biosciences). Flow cytometry was analyzed using FlowJo software (Version 10; BD Biosciences, Ashland, OR, USA).

Single-cell suspensions were labeled with fluorophore-conjugated anti-mouse antibodies (Table 1) and Zombie viability dye at recommended dilutions following the manufacturers’ recommendations (BioLegend). Data were acquired on a BD LSR II using FACS DIVA software with automated compensation (BD Biosciences). Compensation data were acquired using single stained BD Comp beads as per manufacturer’s instructions (BD Biosciences). All data were analyzed using FlowJo software (BD Biosciences).